Transition metal complexes with bidentate ligand having an imidazole ring

ABSTRACT

Novel transition metal complexes of iron, cobalt, ruthenium, osmium, and vanadium are described. The transition metal complexes can be used as redox mediators in enzyme based electrochemical sensors. In such instances, transition metal complexes accept electrons from, or transfer electrons to, enzymes at a high rate and also exchange electrons rapidly with the sensor. The transition metal complexes include at least one substituted or unsubstituted biimidazole ligand and may further include a second substituted or unsubstituted biimidazole ligand or a substituted or unsubstituted bipyridine or pyridylimidazole ligand. Transition metal complexes attached to polymeric backbones are also described.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/712,452, filed Nov. 14, 2000, issued as U.S. Pat. No. 6,605,201,which is a continuation-in-part of U.S. Provisional Patent ApplicationSer. No. 60/165,565, filed Nov. 15, 1999, which are incorporated hereinby reference.

FIELD OF THE INVENTION

This invention relates to transition metal complexes with at least onebidentate ligand containing at least one imidazole ring. In addition,the invention relates to the preparation of the transition metalcomplexes and to the use of the transition metal complexes as redoxmediators.

BACKGROUND OF THE INVENTION

Enzyme based electrochemical sensors are widely used in the detection ofanalytes in clinical, environmental, agricultural and biotechnologicalapplications. Analytes that can be measured in clinical assays of fluidsof the human body include, for example, glucose, lactate, cholesterol,bilirubin and amino acids. Levels of these analytes in biologicalfluids, such as blood, are important for the diagnosis and themonitoring of diseases.

Electrochemical assays are typically performed in cells with two orthree electrodes, including at least one measuring or working electrodeand one reference electrode. In three electrode systems, the thirdelectrode is a counter-electrode. In two electrode systems, thereference electrode also serves as the counter-electrode. The electrodesare connected through a circuit, such as a potentiostat. The measuringor working electrode is a non-corroding carbon or metal conductor. Uponpassage of a current through the working electrode, a redox enzyme iselectrooxidized or electroreduced. The enzyme is specific to the analyteto be detected, or to a product of the analyte. The turnover rate of theenzyme is typically related (preferably, but not necessarily, linearly)to the concentration of the analyte itself, or to its product, in thetest solution.

The electrooxidation or electroreduction of the enzyme is oftenfacilitated by the presence of a redox mediator in the solution or onthe electrode. The redox mediator assists in the electricalcommunication between the working electrode and the enzyme. The redoxmediator can be dissolved in the fluid to be analyzed, which is inelectrolytic contact with the electrodes, or can be applied within acoating on the working electrode in electrolytic contact with theanalyzed solution. The coating is preferably not soluble in water,though it may swell in water. Useful devices can be made, for example,by coating an electrode with a film that includes a redox mediator andan enzyme where the enzyme is catalytically specific to the desiredanalyte, or its product. In contrast to a coated redox mediator, adiffusional redox mediator, which can be soluble or insoluble in water,functions by shuttling electrons between, for example, the enzyme andthe electrode. In any case, when the substrate of the enzyme iselectrooxidized, the redox mediator transports electrons from thesubstrate-reduced enzyme to the electrode; when the substrate iselectroreduced, the redox mediator transports electrons from theelectrode to the substrate-oxidized enzyme.

Recent enzyme based electrochemical sensors have employed a number ofdifferent redox mediators such as monomeric ferrocenes,quinoid-compounds including quinines (e.g., benzoquinones), nickelcyclamates, and ruthenium ammines. For the most part, these redoxmediators have one or more of the following limitations: the solubilityof the redox mediators in the test solutions is low, their chemical,light, thermal, or pH stability is poor, or they do not exchangeelectrons rapidly enough with the enzyme or the electrode or both.Additionally, the redox potentials of many of these reported redoxmediators are so oxidizing that at the potential where the reducedmediator is electrooxidized on the electrode, solution components otherthan the analyte are also electrooxidized; in other cases they are soreducing that solution components, such as, for example, dissolvedoxygen are also rapidly electroreduced. As a result, the sensorutilizing the mediator is not sufficiently specific.

SUMMARY OF THE INVENTION

The present invention is directed to novel transition metal complexes.The present invention is also directed to the use of the complexes asredox mediators. The preferred redox mediators typically exchangeelectrons rapidly with enzymes and electrodes, are stable, and have aredox potential that is tailored for the electrooxidation of analytes,exemplified by glucose.

One embodiment of the invention is a transition metal complex having theformula:

M is cobalt, ruthenium, osmium, or vanadium. L is selected from thegroup consisting of:

R₁, R₂, and R′₁ are independently substituted or unsubstituted alkyl,alkenyl, or aryl groups. R₃, R₄, R₅, R₆, R′₃, R′₄, R_(a), R_(b), R_(c),and R_(d) are independently —H, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H,—SO₃H, —NHNH₂, —SH, aryl, alkoxycarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, —OH, alkoxy, —NH₂, alkylamino, dialkylamino,alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl. c is aninteger selected from −1 to −5 or +1 to +5 indicating a positive ornegative charge. X represents at least one counter ion and d is aninteger from 1 to 5 representing the number of counter ions, X. L₁, L₂,L₃ and L₄ are other ligands.

Another embodiment is a redox mediator having the formula:

M is iron, cobalt, ruthenium, osmium, or vanadium. L is a bidentateligand comprising at least one imidazole ring. c is an integer selectedfrom −1 to −5 or +1 to +5 indicating a positive or negative charge. Xrepresents at least one counter ion and d is an integer from 1 to 5representing the number of counter ions, X. L₁, L₂, L₃ and L₄ are otherligands.

Another embodiment is a sensor that includes the redox polymer, aworking electrode, and a counter electrode. The redox polymer isdisposed proximate to the working electrode.

Yet another embodiment is a polymer that includes a polymeric backboneand a transition metal complex having the following formula:

M is iron, cobalt, ruthenium, osmium, or vanadium. L is a bidentateligand comprising at least one imidazole ring. c is an integer selectedfrom −1 to −5 or +1 to +5 indicating a positive or negative charge. Xrepresents at least one counter ion and d is an integer from 1 to 5representing the number of counter ions, X. L₁, L₂, L₃ and L₄ are otherligands where at least one of L, L₁, L₂, L₃ and L₄ couples to thepolymeric backbone.

DETAILED DESCRIPTION

When used herein, the following definitions define the stated term:

The term “alkyl” includes linear or branched, saturated aliphatichydrocarbons. Examples of alkyl groups include methyl, ethyl, n-propyl,isopropyl, n-butyl, tert-butyl and the like. Unless otherwise noted, theterm “alkyl” includes both alkyl and cycloalkyl groups.

The term “alkoxy” describes an alkyl group joined to the remainder ofthe structure by an oxygen atom. Examples of alkoxy groups includemethoxy, ethoxy, n-propoxy, isopropoxy, butoxy, tert-butoxy, and thelike. In addition, unless otherwise noted, the term ‘alkoxy’ includesboth alkoxy and cycloalkoxy groups.

The term “alkenyl” describes an unsaturated, linear or branchedaliphatic hydrocarbon having at least one carbon-carbon double bond.Examples of alkenyl groups include ethenyl, 1-propenyl, 2-propenyl,1-butenyl, 2-methyl-1-propenyl, and the like.

A “reactive group” is a functional group of a molecule that is capableof reacting with another compound to couple at least a portion of thatother compound to the molecule. Reactive groups include carboxy,activated ester, sulfonyl halide, sulfonate ester, isocyanate,isothiocyanate, epoxide, aziridine, halide, aldehyde, ketone, amine,acrylamide, thiol, acyl azide, acyl halide, hydrazine, hydroxylamine,alkyl halide, imidazole, pyridine, phenol, alkyl sulfonate,halotriazine, imido ester, maleimide, hydrazide, hydroxy, andphoto-reactive azido aryl groups. Activated esters, as understood in theart, generally include esters of succinimidyl, benzotriazolyl, or arylsubstituted by electron-withdrawing groups such as sulfo, nitro, cyano,or halo groups; or carboxylic acids activated by carbodiimides.

A “substituted” functional group (e.g., substituted alkyl, alkenyl, oralkoxy group) includes at least one substituent selected from thefollowing: halogen, alkoxy, mercapto, aryl, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, —OH, —NH₂, alkylamino,dialkylamino, trialkylammonium, alkanoylamino, arylcarboxamido,hydrazino, alkylthio, alkenyl, and reactive groups.

A “biological fluid” is any body fluid or body fluid derivative in whichthe analyte can be measured, for example, blood, interstitial fluid,plasma, dermal fluid, sweat, and tears.

An “electrochemical sensor” is a device configured to detect thepresence of or measure the concentration or amount of an analyte in asample via electrochemical oxidation or reduction reactions. Thesereactions typically can be transduced to an electrical signal that canbe correlated to an amount or concentration of analyte.

A “redox mediator” is an electron transfer agent for carrying electronsbetween an analyte or an analyte-reduced or analyte-oxidized enzyme andan electrode, either directly, or via one or more additional electrontransfer agents.

“Electrolysis” is the electrooxidation or electroreduction of a compoundeither directly at an electrode or via one or more electron transferagents (e.g., redox mediators or enzymes).

The term “reference electrode” includes both a) reference electrodes andb) reference electrodes that also function as counter electrodes (i.e.,counter/reference electrodes), unless otherwise indicated.

The term “counter electrode” includes both a) counter electrodes and b)counter electrodes that also function as reference electrodes (i.e.,counter/reference electrodes), unless otherwise indicated.

Generally, the present invention relates to transition metal complexesof iron, cobalt, ruthenium, osmium, and vanadium having at least onebidentate ligand containing an imidazole ring. The invention alsorelates to the preparation of the transition metal complexes and to theuse of the transition metal complexes as redox mediators. In at leastsome instances, the transition metal complexes have one or more of thefollowing characteristics: redox potentials in a particular range, theability to exchange electrons rapidly with electrodes, the ability torapidly transfer electrons to or rapidly accept electrons from an enzymeto accelerate the kinetics of electrooxidation or electroreduction of ananalyte in the presence of an enzyme or another analyte-specific redoxcatalyst. For example, a redox mediator may accelerate theelectrooxidation of glucose in the presence of glucose oxidase orPQQ-glucose dehydrogenase, a process that can be useful for theselective assay of glucose in the presence of other electrochemicallyoxidizable species. Compounds having the formula 1 are examples oftransition metal complexes of the present invention.

M is a transition metal and is typically iron, cobalt, ruthenium,osmium, or vanadium. Ruthenium and osmium are particularly suitable forredox mediators.

L is a bidentate ligand containing at least one imidazole ring. Oneexample of L is a 2,2′-biimidazole having the following structure 2:

R₁ and R₂ are substituents attached to two of the 2,2′-biimidazolenitrogens and are independently substituted or unsubstituted alkyl,alkenyl, or aryl groups. Generally, R₁ and R₂ are unsubstituted C1 toC12 alkyls. Typically, R₁ and R₂ are unsubstituted C1 to C4 alkyls. Insome embodiments, both R₁ and R₂ are methyl.

R₃, R₄, R₅, and R₆ are substituents attached to carbon atoms of the2,2′-biimidazole and are independently —H, —F, —Cl, —Br, —I, —NO₂, —CN,—CO₂H, —SO₃H, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,—OH, alkoxy, —NH₂, alkylamino, dialkylamino, alkanoylamino,arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino,alkylthio, alkenyl, aryl, or alkyl. Alternatively, R₃ and R₄ incombination or R₅ and R₆ in combination independently form a saturatedor unsaturated 5- or 6-membered ring. An example of this is a2,2′-bibenzoimidazole derivative. Typically, the alkyl and alkoxyportions are C1 to C12. The alkyl or aryl portions of any of thesubstituents are optionally substituted by —F, —Cl, —Br, —I, alkylamino,dialkylamino, trialkylammonium (except on aryl portions), alkoxy,alkylthio, aryl, or a reactive group. Generally, R₃, R₄, R₅, and R₆ areindependently —H or unsubstituted alkyl groups. Typically, R₃, R₄, R₅,and R₆ are —H or unsubstituted C1 to C12 alkyls. In some embodiments,R₃, R₄, R₅, and R₆ are all —H.

Another example of L is a 2-(2-pyridyl)imidazole having the followingstructure 3:

R′₁ is a substituted or unsubstituted aryl, alkenyl, or alkyl.Generally, R′₁ is a substituted or unsubstituted C1-C12 alkyl. R′₁ istypically methyl or a C1-C12 alkyl that is optionally substituted with areactive group.

R′₃, R′₄, R_(a), R_(b), R_(c), and R_(d) are independently —H, —F, —Cl,—Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, alkoxylcarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH₂, alkylamino,dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl, or alkyl.Alternatively, R_(c) and R_(d) in combination or R′₃ and R′₄ incombination can form a saturated or unsaturated 5- or 6-membered ring.Typically, the alkyl and alkoxy portions are C1 to C12. The alkyl oraryl portions of any of the substituents are optionally substituted by—F, —Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except onaryl portions), alkoxy, alkylthio, aryl, or a reactive group. Generally,R′₃, R′₄, R_(a), R_(b), R_(c) and R_(d) are independently —H orunsubstituted alkyl groups. Typically, R_(a) and R_(c) are —H and R′₃,R′₄, R_(b), and R_(d) are —H or methyl.

c is an integer indicating the charge of the complex. Generally, c is aninteger selected from −1 to −5 or +1 to +5 indicating a positive ornegative charge. For a number of osmium complexes, c is +2 or +3.

X represents counter ion(s). Examples of suitable counter ions includeanions, such as halide (e.g., fluoride, chloride, bromide or iodide),sulfate, phosphate, hexafluorophosphate, and tetrafluoroborate, andcations (preferably, monovalent cations), such as lithium, sodium,potassium, tetralkylammonium, and ammonium. Preferably, X is a halide,such as chloride. The counter ions represented by X are not necessarilyall the same.

d represents the number of counter ions and is typically from 1 to 5.

L₁, L₂, L₃ and L₄ are ligands attached to the transition metal via acoordinative bond. L₁, L₂, L₃ and L₄ can be monodentate ligands or, inany combination, bi-, ter-, or tetradentate ligands For example, L₁, L₂,L₃ and L₄ can combine to form two bidentate ligands such as, forexample, two ligands selected from the group of substituted andunsubstituted 2,2′-biimidazoles, 2-(2-pyridyl)imidizoles, and2,2′-bipyridines

Examples of other L₁, L₂, L₃ and L₄ combinations of the transition metalcomplex include:

-   -   (A) L₁ is a monodentate ligand and L₂, L₃ and L₄ in combination        form a terdentate ligand;    -   (B) L₁ and L₂ in combination are a bidentate ligand, and L₃ and        L₄ are the same or different monodentate ligands;    -   (C) L₁ and L₂ in combination, and L₃ and L₄ in combination form        two independent bidentate ligands which can be the same or        different; and    -   (D) L₁, L₂, L₃ and L₄ in combination form a tetradentate ligand.

Examples of suitable monodentate ligands include, but are not limitedto, —F, —Cl, —Br, —I, —CN, —SCN, —OH, H₂O, NH₃, alkylamine,dialkylamine, trialkylamine, alkoxy or heterocyclic compounds. The alkylor aryl portions of any of the ligands are optionally substituted by —F,—Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except on arylportions), alkoxy, alkylthio, aryl, or a reactive group. Any alkylportions of the monodentate ligands generally contain 1 to 12 carbons.More typically, the alkyl portions contain 1 to 6 carbons. In otherembodiments, the monodentate ligands are heterocyclic compoundscontaining at least one nitrogen, oxygen, or sulfur atom. Examples ofsuitable heterocyclic monodentate ligands include imidazole, pyrazole,oxazole, thiazole, pyridine, pyrazine and derivatives thereof. Suitableheterocyclic monodentate ligands include substituted and unsubstitutedimidazole and substituted and unsubstituted pyridine having thefollowing general formulas 4 and 5, respectively:

With regard to formula 4, R₇ is generally a substituted or unsubstitutedalkyl, alkenyl, or aryl group. Typically, R₇ is a substituted orunsubstituted C1 to C12 alkyl or alkenyl. The substitution of innercoordination sphere chloride anions by imidazoles does not typicallycause a large shift in the redox potential in the oxidizing direction,differing in this respect from substitution by pyridines, whichtypically results in a large shift in the redox potential in theoxidizing direction.

R₈, R₉ and R₁₀ are independently —H, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H,—SO₃H, —NHNH₂, —SH, aryl, alkoxycarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, —OH, alkoxy, —NH₂, alkylamino, dialkylamino,alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl.Alternatively, R₉ and R₁₀, in combination, form a fused 5 or 6-memberedring that is saturated or unsaturated. The alkyl portions of thesubstituents generally contain 1 to 12 carbons and typically contain 1to 6 carbon atoms. The alkyl or aryl portions of any of the substituentsare optionally substituted by —F, —Cl, —Br, —I, alkylamino,dialkylamino, trialkylammonium (except on aryl portions), alkoxy,alkylthio, aryl, or a reactive group. In some embodiments, R₈, R₉ andR₁₀ are —H or substituted or unsubstituted alkyl. Preferably, R₈, R₉ andR₁₀ are —H.

With regard to Formula 5, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ are independently—H, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH₂, alkylamino,dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl. Thealkyl or aryl portions of any of the substituents are optionallysubstituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino,trialkylammonium (except for aryl portions), alkoxy, alkylthio, aryl, ora reactive group. Generally, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ are —H, methyl,C1-C2 alkoxy, C1-C2 alkylamino, C2-C4 dialkylamino, or a C1-C6 loweralkyl substituted with a reactive group.

One example includes R₁₁ and R₁₅ as —H, R₁₂ and R₁₄ as the same and —Hor methyl, and R₁₃ as —H, C1 to C12 alkoxy, —NH₂, C1 to C12 alkylamino,C2 to C24 dialkylamino, hydrazino, C1 to C12 alkylhydrazino,hydroxylamino, C1 to C12 alkoxyamino, C1 to C12 alkylthio, or C1 to C12alkyl. The alkyl or aryl portions of any of the substituents areoptionally substituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino,trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, ora reactive group.

Examples of suitable bidentate ligands include, but are not limited to,amino acids, oxalic acid, acetylacetone, diaminoalkanes,ortho-diaminoarenes, 2,2′-biimidazole, 2,2′-bioxazole, 2,2′-bithiazole,2-(2-pyridyl)imidazole, and 2,2′-bipyridine and derivatives thereof.Particularly suitable bidentate ligands for redox mediators includesubstituted and unsubstituted 2,2′-biimidazole, 2-(2-pyridyl)imidazoleand 2,2′-bipyridine. The substituted 2,2′ biimidazole and2-(2-pyridyl)imidazole ligands can have the same substitution patternsdescribed above for the other 2,2′-biimidazole and2-(2-pyridyl)imidazole ligand. A 2,2′-bipyridine ligand has thefollowing general formula 6:

R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, R₂₁, R₂₂ and R₂₃ are independently —H, —F, —Cl,—Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, aryl, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH₂, alkylamino,dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxylamino, alkylthio, alkenyl, or alkyl. Typically,the alkyl and alkoxy portions are C1 to C12. The alkyl or aryl portionsof any of the substituents are optionally substituted by —F, —Cl, —Br,—I, alkylamino, dialkylamino, trialkylammonium (except on arylportions), alkoxy, alkylthio, aryl, or a reactive group.

Specific examples of suitable combinations of R₁₆, R₁₇, R₁₈, R₁₉, R₂₀,R₂₁, R₂₂ and R₂₃ include R₁₆ and R₂₃ as H or methyl; R₁₇ and R₂₂ as thesame and —H or methyl; and R₁₉ and R₂₀ as the same and —H or methyl. Analternative combination is where one or more adjacent pairs ofsubstituents R₁₆ and R₁₇, on the one hand, and R₂₂ and R₂₃, on the otherhand, independently form a saturated or unsaturated 5- or 6-memberedring. Another combination includes R₁₉ and R₂₀ forming a saturated orunsaturated five or six membered ring.

Another combination includes R₁₆, R₁₇, R₁₉, R₂₀, R₂₂ and R₂₃ as the sameand —H and R₁₈ and R₂₁ as independently —H, alkoxy, —NH₂, alkylamino,dialkylamino, alkylthio, alkenyl, or alkyl. The alkyl or aryl portionsof any of the substituents are optionally substituted by —F, —Cl, —Br,—I, alkylamino, dialkylamino, trialkylammonium (except on arylportions), alkoxy, alkylthio, aryl, or a reactive group. As an example,R₁₈ and R₂₁ can be the same or different and are —H, C1-C6 alkyl, C1-C6amino, C1 to C12 alkylamino, C2 to C12 dialkylamino, C1 to C12alkylthio, or C1 to C12 alkoxy, the alkyl portions of any of thesubstituents are optionally substituted by a —F, —Cl, —Br, —I, aryl, C2to C12 dialkylamino, C3 to C18 trialkylammonium, C1 to C6 alkoxy, C1 toC6 alkylthio or a reactive group.

Examples of suitable terdentate ligands include, but are not limited to,diethylenetriamine, 2,2′,2″-terpyridine, 2,6-bis(N-pyrazolyl)pyridine,and derivatives of these compounds. 2,2′,2″-terpyridine and2,6-bis(N-pyrazolyl)pyridine have the following general formulas 7 and 8respectively:

With regard to formula 7, R₂₄, R₂₅ and R₂₆ are independently —H orsubstituted or unsubstituted C1 to C12 alkyl. Typically, R₂₄, R₂₅ andR₂₆ are —H or methyl and, in some embodiments, R₂₄ and R₂₆ are the sameand are —H. Other substituents at these or other positions of thecompounds of formulas 7 and 8 can be added.

With regard to formula 8, R₂₇, R₂₈ and R₂₉ are independently —H, —F,—Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH₂, alkylamino,dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl, or alkyl. Thealkyl or aryl portions of any of the substituents are optionallysubstituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino,trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, ora reactive group. Typically, the alkyl and alkoxy groups are C1 to C12and, in some embodiments, R₂₇ and R₂₉ are the same and are —H.

Examples of suitable tetradentate ligands include, but are not limitedto, triethylenetriamine, ethylenediaminediacetic acid, tetraazamacrocycles and similar compounds as well as derivatives thereof.

Examples of suitable transition metal complexes are illustrated usingFormula 9 and 10:

With regard to transition metal complexes of formula 9, the metal osmiumis complexed to two substituted 2,2′-biimidazole ligands and onesubstituted or unsubstituted 2,2′-bipyridine ligand. R₁, R₂, R₃, R₄, R₅,R₆, R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, R₂₁, R₂₂, R₂₃, c, d, and X are the same asdescribed above.

In one embodiment, R₁ and R₂ are methyl; R₃, R₄, R₅, R₆, R₁₆, R₁₇, R₁₉,R₂₀, R₂₂ and R₂₃ are —H; and R₁₈ and R₂₁ are the same and are —H,methyl, or methoxy. Preferably, R₁₈ and R₂₁ are methyl or methoxy.

In another embodiment, R₁ and R₂ are methyl; R₃, R₄, R₅, R₆, R₁₆, R₁₇,R₁₈, R₁₉, R₂₀, R₂₂ and R₂₃ are —H; and R₂₁ is halo, C1 to C12 alkoxy, C1to C12 alkylamino, or C2 to C24 dialkylamino. The alkyl or aryl portionsof any of the substituents are optionally substituted by —F, —Cl, —Br,—I, alkylamino, dialkylamino, trialkylammonium (except on arylportions), alkoxy, alkylthio, aryl, or a reactive group. For example,R₂₁ is a C1 to C12 alkylamino or C2 to C24 dialkylamino, the alkylportion(s) of which are substituted with a reactive group, such as acarboxylic acid, activated ester, or amine. Typically, the alkylaminogroup has 1 to 6 carbon atoms and the dialkylamino group has 2 to 8carbon atoms.

With regard to transition metal complexes of formula 10, the metalosmium is complexed to two substituted 2,2′-biimidazole ligands and onesubstituted or unsubstituted 2-(2-pyridyl)imidazole ligand. R₁, R₂, R₃,R₄, R₅, R₆, R′₁, R′₃, R′₄, R_(a), R_(b), R_(c), R_(d), c, d, and X arethe same as described above.

In one embodiment, R₁ and R₂ are methyl; R₃, R₄, R₅, R₆, R′₃, R′₄ andR_(d) are independently —H or methyl; R_(a) and R_(c) are the same andare —H; and R_(b) is C1 to C12 alkoxy, C1 to C12 alkylamino, or C2 toC24 dialkylamino. The alkyl or aryl portions of any of the substituentsare optionally substituted by —F, —Cl, —Br, —I, alkylamino,dialkylamino, trialkylammonium (except on aryl portions), alkoxy,alkylthio, aryl, or a reactive group.

A list of specific examples of preferred transition metal complexes withrespective redox potentials is shown in Table 1. TABLE 1 RedoxPotentials of Selected Transition Metal Complexes Complex StructureE_(½)(vs Ag/AgCl)/mV* I

−110 II

−100 III

128 IV

−86 V

−97 VI

−120 VII

32 VIII

−100 IX

−93 X

−125 XI

−60 XII

−74 XIII

−97 IVX

−81 VX

−230*Redox potentials were estimated by averaging the positions of thereduction wave peaks and the oxidation wave peaks of cyclicvoltammograms (CVs) obtained in pH 7 PBS buffer with a glassy carbonworking electrode, a graphite counter electrode and a standard Ag/AgClreference electrode at a sweep rate of 50 mV/s.

The transition metal complexes of Formula 1 also include transitionmetal complexes that are coupled to a polymeric backbone through one ormore of L, L₁, L₂, L₃, and L₄. Additional examples of suitabletransition metal complexes are described in U.S. patent application Ser.No. 09/712,065, entitled “Polymeric Transition Metal Complexes and UsesThereof”, filed on even date herewith, incorporated herein by reference.In some embodiments, the polymeric backbone has functional groups thatact as ligands of the transitional metal complex. Such polymericbackbones include, for example, poly(4-vinylpyridine) andpoly(N-vinylimidazole) in which the pyridine and imidazole groups,respectively, can act as monodentate ligands of the transition metalcomplex. In other embodiments, the transition metal complex can be thereaction product between a reactive group on a precursor polymer and areactive group on a ligand of a precursor transition metal complex (suchas a complex of Formula 1 where one of L, L₁, L₂, L₃ and L₄ includes areactive group as described above). Suitable precursor polymers include,for example, poly(acrylic acid) (Formula 11), styrene/maleic anhydridecopolymer (Formula 12), methylvinylether/maleic anhydride copolymer(GANTREX polymer) (Formula 13), poly(vinylbenzylchloride) (Formula 14),poly(allylamine) (Formula 15), polylysine (Formula 16),carboxy-poly(vinylpyridine (Formula 17), and poly(sodium 4-styrenesulfonate) (Formula 18).

Alternatively, the transition metal complex can have reactive group(s)for immobilization or conjugation of the complexes to other substratesor carriers, examples of which include, but are not limited to,macromolecules (e.g., enzymes) and surfaces (e.g., electrode surfaces).

For reactive attachment to polymers, substrates, or other carriers, thetransition metal complex precursor includes at least one reactive groupthat reacts with a reactive group on the polymer, substrate, or carrier.Typically, covalent bonds are formed between the two reactive groups togenerate a linkage. Examples of such linkages are provided in Table 2,below. Generally, one of the reactive groups is an electrophile and theother reactive group is a nucleophile. TABLE 2 Examples of ReactiveGroup Linkages First Reactive Group Second Reactive Group ResultingLinkage Activated ester* Amine Carboxamide Acrylamide Thiol ThioetherAcyl azide Amine Carboxamide Acyl halide Amine Carboxamide Carboxylicacid Amine Carboxamide Aldehyde or ketone Hydrazine Hydrazone Aldehydeor ketone Hydroxyamine Oxime Alkyl halide Amine Alkylamine Alkyl halideCarboxylic acid Carboxylic ester Alkyl halide Imidazole ImidazoliumAlkyl halide Pyridine Pyridinium Alkyl halide Alcohol/phenol Ether Alkylhalide Thiol Thioether Alkyl sulfonate Thiol Thioether Alkyl sulfonatePyridine Pyridinium Alkyl sulfonate Imidazole Imidazolium Alkylsulfonate Alcohol/phenol Ether Anhydride Alcohol/phenol Ester AnhydrideAmine Carboxamide Aziridine Thiol Thioether Aziridine Amine AlkylamineAziridine Pyridine Pyridinium Epoxide Thiol Thioether Epoxide AmineAlkylamine Epoxide Pyridine Pyridinium Halotriazine Amine AminotriazineHalotriazine Alcohol Triazinyl ether Imido ester Amine AmidineIsocyanate Amine Urea Isocyanate Alcohol Urethane Isothiocyanate AmineThiourea Maleimide Thiol Thioether Sulfonyl halide Amine Sulfonamide*Activated esters, as understood in the art, generally include esters ofsuccinimidyl, benzotriazolyl, or aryl substituted byelectron-withdrawing groups such as sulfo, nitro, cyano, or halo; orcarboxylic acids activated by carbodiimides.

Transition metal complexes of the present invention can be soluble inwater or other aqueous solutions, or in organic solvents. In general,the transition metal complexes can be made soluble in either aqueous ororganic solvents by having an appropriate counter ion or ions, X. Forexample, transition metal complexes with small counter anions, such asF⁻, Cl⁻, and Br⁻, tend to be water soluble. On the other hand,transition metal complexes with bulky counter anions, such as I⁻, BF₄ ⁻and PF₆ ⁻, tend to be soluble in organic solvents. Preferably, thesolubility of transition metal complexes of the present invention isgreater than about 0.1 M (moles/liter) at 25° C. for a desired solvent.

The transition metal complexes discussed above are useful as redoxmediators in electrochemical sensors for the detection of analytes inbio-fluids. The use of transition metal complexes as redox mediators isdescribed, for example, in U.S. Pat. Nos. 5,262,035; 5,262,305;5,320,725; 5,365,786; 5,593,852; 5,665,222; 5,972,199; and 6,143,164 andU.S. patent applications Ser. Nos. 09/034,372, (now U.S. Pat. No.6,134,461); Ser. No. 09/070,677, (now U.S. Pat. No. 6,175,752); Ser. No.09/295,962, (now U.S. Pat. No. 6,338,790) and Ser. No. 09/434,026, allof which are herein incorporated by reference. The transitional metalcomplexes described herein can typically be used in place of thosediscussed in the references listed above. The transitions metalcomplexes that include a polymeric backbone and are redox mediators canalso be referred to as “redox polymers

In general, the redox mediator is disposed on or in proximity to (e.g.,in a solution surrounding) a working electrode. The redox mediatortransfers electrons between the working electrode and an analyte. Insome preferred embodiments, an enzyme is also included to facilitate thetransfer. For example, the redox mediator transfers electrons betweenthe working electrode and glucose (typically via an enzyme) in anenzyme-catalyzed reaction of glucose. Redox polymers are particularlyuseful for forming non-leachable coatings on the working electrode.These can be formed, for example, by crosslinking the redox polymer onthe working electrode, or by crosslinking the redox polymer and theenzyme on the working electrode

Transition metal complexes can enable accurate, reproducible and quickor continuous assays. Transition metal complex redox mediators acceptelectrons from, or transfer electrons to, enzymes or analytes at a highrate and also exchange electrons rapidly with an electrode. Typically,the rate of self exchange, the process in which a reduced redox mediatortransfers an electron to an oxidized redox mediator, is rapid. At adefined redox mediator concentration, this provides for more rapidtransport of electrons between the enzyme (or analyte) and electrode,and thereby shortens the response time of the sensor. Additionally, thenovel transition metal complex redox mediators are typically stableunder ambient light and at the temperatures encountered in use, storageand transportation. Preferably, the transition metal complex redoxmediators do not undergo chemical change, other than oxidation andreduction, in the period of use or under the conditions of storage,though the redox mediators can be designed to be activated by reacting,for example, with water or the analyte.

The transition metal complex can be used as a redox mediator incombination with a redox enzyme to electrooxidize or electroreduce theanalyte or a compound derived of the analyte, for example by hydrolysisof the analyte. The redox potentials of the redox mediators aregenerally more positive (i.e. more oxidizing) than the redox potentialsof the redox enzymes when the analyte is electrooxidized and morenegative when the analyte is electroreduced. For example, the redoxpotentials of the preferred transition metal complex redox mediatorsused for electrooxidizing glucose with glucose oxidase or PQQ-glucosedehydrogenase as enzyme is between about −200 mV and +200 mV versus aAg/AgCl reference electrode, and the most preferred mediators have redoxpotentials between about −100 mV and about +100 mV versus a Ag/AgClreference electrode

Crosslinking in Transition Metal Complex Polymers

Electron transport involves an exchange of electrons between segments ofthe redox polymers (e.g., one or more transition metal complexes coupledto a polymeric backbone, as described above) in a crosslinked filmdisposed on an electrode. The transition metal complex can be bound tothe polymer backbone though covalent, coordinative or ionic bonds, wherecovalent and coordinative binding are preferred. Electron exchangeoccurs, for example, through the collision of different segments of thecrosslinked redox polymer. Electrons transported through the redoxpolymer can originate from, for example, electrooxidation orelectroreduction of an enzymatic substrate, such as, for example, theoxidation of glucose by glucose oxidase.

The degree of crosslinking of the redox polymer can influence thetransport of electrons or ions and thereby the rates of theelectrochemical reactions. Excessive crosslinking of the polymer canreduce the mobility of the segments of the redox polymer. A reduction insegment mobility can slow the diffusion of electrons or ions through theredox polymer film. A reduction in the diffusivity of electrons, forexample, can require a concomitant reduction in the thickness of thefilm on the electrode where electrons or electron vacancies arecollected or delivered. The degree of crosslinking in a redox polymerfilm can thus affect the transport of electrons from, for example, anenzyme to the transition metal redox centers of the redox polymer suchas, for example, Os^(2+/3+) metal redox centers; between redox centersof the redox polymer; and from these transition metal redox centers tothe electrode.

Inadequate crosslinking of a redox polymer can result in excessiveswelling of the redox polymer film and to the leaching of the componentsof the redox polymer film. Excessive swelling can also result in themigration of the swollen polymer into the analyzed solution, in thesoftening of the redox polymer film, in the film's susceptibility toremoval by shear, or any combination of these effects.

Crosslinking can decrease the leaching of film components and canimprove the mechanical stability of the film under shear stress. Forexample, as disclosed in Binyamin, G. and Heller, A; Stabilization ofWired Glucose Oxidase Anodes Rotating at 1000 rpm at 37° C.; Journal ofthe Electrochemical Society, 146(8), 2965-2967, 1999, hereinincorporated by reference, replacing a difunctional crosslinker, such aspolyethylene glycol diglycidyl ether, with a trifunctional crosslinkersuch as N,N-diglycidyl-4-glycidyloxyaniline, for example, can reduceleaching and shear problems associated with inadequate crosslinking.

Examples of other bifunctional, trifunctional and tetrafunctionalcrosslinkers are listed below:

Alternatively, the number of crosslinking sites can be increased byreducing the number of transition metal complexes attached to thepolymeric backbone, thus making more polymer pendant groups availablefor crosslinking. One important advantage of at least some of the redoxpolymers is the increased mobility of the pendant transition metalcomplexes, resulting from the flexibility of the pendant groups. As aresult, in at least some embodiments, fewer transition metal complexesper polymer backbone are needed to achieve a desired level ofdiffusivity of electrons and current density of analyte electrooxidationor electroreduction.

Coordination in Transition Metal Complex Polymers

Transition metal complexes can be directly or indirectly attached to apolymeric backbone, depending on the availability and nature of thereactive groups on the complex and the polymeric backbone. For example,the pyridine groups in poly(4-vinylpyridine) or the imidazole groups inpoly(N-vinylimidazole) are capable of acting as monodentate ligands andthus can be attached to a metal center directly. Alternatively, thepyridine groups in poly(4-vinylpyridine) or the imidazole groups inpoly(N-vinylimidazole) can be quaternized with a substituted alkylmoiety having a suitable reactive group, such as a carboxylate function,that can be activated to form a covalent bond with a reactive group,such as an amine, of the transition metal complex. (See Table 2 for alist of other examples of reactive groups.)

Redox centers such as, for example, Os^(2+/3+) can be coordinated withfive heterocyclic nitrogens and an additional ligand such as, forexample, a chloride anion. An example of such a coordination complexincludes two bipyridine ligands which form stable coordinative bonds,the pyridine of poly(4-vinylpyridine) which forms a weaker coordinativebond, and a chloride anion which forms the least stable coordinativebond.

Alternatively, redox centers, such as Os^(2+/3+), can be coordinatedwith six heterocyclic nitrogen atoms in its inner coordination sphere.The six coordinating atoms are preferably paired in the ligands, forexample, each ligand is composed of at least two rings. Pairing of thecoordinating atoms can influence the potential of an electrode used inconjunction with redox polymers of the present invention.

Typically, for analysis of glucose, the potential at which the workingelectrode, coated with the redox polymer, is poised is negative of about±250 mV vs. SCE (standard calomel electrode). Preferably, the electrodeis poised negative of about +150 mV vs. SCE. Poising the electrode atthese potentials reduces the interfering electrooxidation ofconstituents of biological solutions such as, for example, urate,ascorbate and acetaminophen. The potential can be modified by alteringthe ligand structure of the complex.

The redox potential of a redox polymer, as described herein, is relatedto the potential at which the electrode is poised. Selection of a redoxpolymer with a desired redox potential allows tuning of the potential atwhich the electrode is best poised. The redox potentials of a number ofthe redox polymers described herein are negative of about +150 mV vs.SCE and can be negative of about +50 mV vs. SCE to allow the poising ofthe electrode potentials negative of about +250 mV vs. SCE andpreferably negative of about +150 mV vs. SCE.

The strength of the coordination bond can influence the potential of theredox centers in the redox polymers. Typically, the stronger thecoordinative bond, the more positive the redox potential. A shift in thepotential of a redox center resulting from a change in the coordinationsphere of the transition metal can produce a labile transition metalcomplex. For example, when the redox potential of an Os^(2+/3+) complexis downshifted by changing the coordination sphere, the complex becomeslabile. Such a labile transition metal complex may be undesirable whenfashioning a metal complex polymer for use as a redox mediator and canbe avoided through the use of weakly coordinating multidentate orchelating heterocyclics as ligands.

Electrode Interference

Transition metal complexes used as redox mediators in electrodes can beaffected by the presence of transition metals in the analyzed sampleincluding, for example, Fe³⁺ or Zn²⁺. The addition of a transition metalcation to a buffer used to test an electrode results in a decline in thecurrent produced. The degree of current decline depends on the presenceof anions in the buffer which precipitate the transition metal cations.The lesser the residual concentration of transition metal cations in thesample solution, the more stable the current. Anions which aid in theprecipitation of transition metal cations include, for example,phosphate. It has been found that a decline in current upon the additionof transition metal cations is most pronounced in non-phosphate buffers.If an electrode is transferred from a buffer containing a transitionmetal cation to a buffer substantially free of the transition metalcation, the original current is restored.

The decline in current is thought to be due to additional crosslinkingof a pyridine-containing polymer backbone produced by the transitionmetal cations. The transition metal cations can coordinate nitrogenatoms of different chains and chain segments of the polymers.Coordinative crosslinking of nitrogen atoms of different chain segmentsby transition metal cations can reduce the diffusivity of electrons.

Serum and other physiological fluids contain traces of transition metalions, which can diffuse into the films of electrodes made with the redoxpolymers of the present invention, lowering the diffusivity of electronsand thereby the highest current reached at high analyte concentration.In addition, transition metal ions like iron and copper can bind toproteins of enzymes and to the reaction centers or channels of enzymes,reducing their turnover rate. The resulting decrease in sensitivity canbe remedied through the use of anions which complex with interferingtransition metal ions, for example, in a buffer employed during theproduction of the transition metal complex. A non-cyclic polyphosphatesuch as, for example, pyrophosphate or triphosphate, can be used. Forexample, sodium or potassium non-cyclic polyphosphate buffers can beused to exchange phosphate anions for those anions in the transitionmetal complex which do not precipitate transition metal ions. The use oflinear phosphates can alleviate the decrease in sensitivity by formingstrong complexes with the damaging transition metal ions, assuring thattheir activity will be low. Other complexing agents can also be used aslong as they are not electrooxidized or electroreduced at the potentialat which the electrode is poised.

Enzyme Damage and its Alleviation

Glucose oxidase is a flavoprotein enzyme that catalyzes the oxidation bydioxygen of D-glucose to D-glucono-1,5-lactone and hydrogen peroxide.Reduced transition metal cations such as, for example, Fe²⁺, and sometransition metal complexes, can react with hydrogen peroxide. Thesereactions form destructive OH radicals and the corresponding oxidizedcations. The presence of these newly formed transition metal cations caninhibit the enzyme and react with the metal complex. Also, the oxidizedtransition metal cation can be reduced by the FADH₂ centers of anenzyme, or by the transition metal complex.

Inhibition of the active site of an enzyme or a transition metal complexby a transition metal cation, as well as damaging reactions with OHradicals can be alleviated, thus increasing the sensitivity andfunctionality of the electrodes by incorporating non-cyclicpolyphosphates, as discussed above. Because the polyphosphate/metalcation complex typically has a high (oxidizing) redox potential, itsrate of oxidation by hydrogen peroxide is usually slow. Alternatively,an enzyme such as, for example, catalase, can be employed to degradehydrogen peroxide.

EXAMPLES

Unless indicated otherwise, all of the chemical reagents are availablefrom Aldrich Chemical Co. (Milwaukee, Wis.) or other sources. Additionalexamples are provided in U.S. Pat. No. 6,605,200 entitled “PolymericTransition Metal Complexes and Uses Thereof”, incorporated herein byreference. For purposes of illustration, the synthesis of severaltransition metal complex ligands are shown below:

Example 1 Synthesis of 4-(5-carboxypentyl)amino-2,2′-bipyridyl

This example illustrates how a carboxy reactive group is introduced ontoa 2.2′-bipyridyl derivative.

Wenkert, D.; Woodward, R. B. J. Org. chem. 48, 283(1983)) (5 g)dissolved in 30 mL acetic acid in a 100 ml round bottom flask was added16 mL acetyl bromide. The yellow mixture was refluxed for 1.5 h and thenrotovaporated to dryness. The resulting light yellow solid of D wassufficiently pure enough for the next step without further purification.Yield: 95%

Synthesis of compound E: To a stirred suspension of compound D in 60 mLCHCl₃ was added 12 mL PCl₃ at room temperature. The mixture was refluxedfor 2 h under N₂ and then cooled to room temperature. The reactionmixture was poured into 100 mL ice/water. The aqueous layer wasseparated and saved. The CHCl₃ layer was extracted three times with H₂O(3×60 mL) and then discarded. The combined aqueous solution wasneutralized with NaHCO₃ powder to about pH 7 to 8. The resulting whiteprecipitate was collected by suction filtration, washed with H₂O (30 mL)and then dried under vacuum at 50° C. for 24 h. Yield: 85%.

Synthesis of compound F: Compound F was synthesized from compound E (5g) and 6-aminocaproic acid methyl ester (6 g) using thepalladium-catalyzed amination method of aryl bromides described byHartwig et al. (Hartwig, J. F., et al. J. Org. Chem. 64, 5575 (1999)).Yield: 90%.

Synthesis of compound G: Compound F (3 g) dissolved in 20 mL MeOH wasadded to a solution of NaOH (0.6 g) in 30 mL H₂O. The resulting solutionwas stirred at room temperature for 24 h and then neutralized to pH 7with dilute HCl. The solution was saturated with NaCl and then extractedwith CHCl₃. The CHCl₃ extract was evaporated to dryness and thenpurified by a silica gel column eluted with 10% H₂O/CH₃CN. Yield: 70%.

Example 2 Synthesis of a 4-((6-Aminohexyl)amino)-2,2′-bipyridine

This example illustrates the general synthesis of a 2,2′-bipyridyl withan amine reactive group.

Synthesis of compound H: A mixture of compound E (2.5 g) and1,6-diaminohexane (15 g) in a 250 mL round bottom flask was heated underN₂ at 140° C. in an oil bath for 4-5 h. Excess 1,6-diaminohexane wasremoved by high vacuum distillation at 90-120° C. The product waspurified by a silica gel column, eluting with 5% NH₄OH in isopropylalcohol. Yield: 70%.

Example 3 Synthesis of 1,1′-dimethyl-2,2′-biimidazole

This example illustrates the synthesis of 2,2′-biimidazole derivatives.

The alkylation step can be carried out stepwise so two different alkylgroups can be introduced. For example:

Synthesis of compound K: To a stirred solution of compound J (formedfrom I according to Fieselmann, B. F., et al. Inorg. Chem. 17,2078(1978)) (4.6 g, 34.3 mmoles) in 100 mL dry DMF in a 250 ml roundbottom flask cooled in an ice/water bath was added in portions NaH(60%in mineral oil, 2.7 g, 68.6 mmoles). After the solution was stirred at0° C. for one more hour under N₂, methyl toluenesulfonate (10.3 mL, 68.6mmoles) was added in small portions using a syringe over 30 min. Thestirring of the solution in the ice/water bath was continued for 1 h andthen at room temperature for 3 h. The solvent was removed by vacuumdistillation. The dark residue was triturated with ether and thensuction filtered and dried under vacuum. The product was purified bysublimation. Yield: 80%.

Synthesis of compound L: Compound L was prepared using the methoddescribed for the synthesis of compound K except that only oneequivalent each of compound J, NaH and methyl toluenesulfonate was used.The product was purified by sublimation.

Synthesis of compound M: To a stirred solution of compound L (1 g, 6.8mmoles) in 20 mL dry DMF in a 50 ml round bottom flask cooled in aice/water bath is added in portions NaH(60% in mineral oil, 0.27 g, 6.8mmoles). After the solution is stirred at 0° C. for one more hour underN₂, ethyl bromoacetate (0.75 mL, 6.8 mmoles) is added in small portionsvia a syringe over 15 min. The stirring of the solution is continued inthe ice/water bath for 1 h and then at room temperature for 3 h. Thesolvent is removed by vacuum distillation. The product is purified by asilica gel column using 10% MeOH/CHCl₃ as the eluent.

Synthesis of Compound N: Compound M (1 g) is hydrolyzed using the methoddescribed for the synthesis of compound G. The product is purified by asilica gel column using 10% H₂O/CH₃CN as the eluent.

Example 4 Synthesis of 2-(2-Pyridyl)imidazole Heterobidentate Ligands

This example illustrates a general synthesis of heterobidentate ligandscontaining an imidazole ring.

Synthesis of compound O: A solution of 6-methylpyridine-2-carboxaldehyde(26 g, 0.21 mole) and glyoxal (40%, 30 mL) in 50 mL EtOH in athree-necked 250 mL round bottom flask fitted with a thermometer and anaddition funnel was stirred in a NaCl/ice bath. When the solution wascooled to below 5° C., conc. NH₄OH was added dropwise through theaddition funnel. The rate of the addition was controlled so that thetemperature of the solution was maintained at below 5° C. After theaddition, the stirring of the yellow solution was continued in the icebath for 1 h and then at room temperature overnight. The light yellowcrystals were collected by suction filtration and washed with H₂O (20mL). The crystals were resuspended in H₂O (200 mL) and boiled briefly,followed by suction filtration, to collect the product which was driedunder high vacuum. Yield: 35%.

Synthesis of compound P: Sodium t-butoxide (2 g, 20.8 mmoles) was addedin one portion to a stirred solution of compound O (3 g, 18.9 mmoles) in50 mL dry DMF. After all of the sodium t-butoxide was dissolved,iodomethane (1.3 mL) was added dropwise using a syringe. The stirring ofthe solution was continued at room temperature for 2 h and then thesolution was poured into H₂O (150 mL). The product was extracted withEtOAc, and the extract was dried with anhydrous Na₂SO₄ and thenevaporated to give crude compound P. The product was purified byseparation on a silica gel column using 10% MeOH/CHCl₃ as the eluent.Yield: 70%.

Example 5 Synthesis of Transition Metal Complexes with MultipleIdentical Ligands

Transition metal complexes containing multiple identical bidentate ortridentate ligands can be synthesized in one step from a metal halidesalt and the ligand. This example illustrates the synthesis of an osmiumcomplex with three identical 2,2′-biimidazole bidentate ligands.

Synthesis of compound Q: Ammonium hexachloroosmate (200 mg, 0.46 mmoles)and compound K (221 mg, 1.37 mmoles) were mixed in 15 mL ethylene glycolin a 100 mL three-necked round bottom flask fitted with a refluxcondenser. The mixture was degassed with N₂ for 15 min and then stirredunder N₂ at 200-210° C. for 24 hrs. The solvent was removed by highvacuum distillation at 90-100° C. The green colored crude product wasdissolved in 15 mL H₂O and stirred in air to be fully oxidized to thedark blue colored Os(III) oxidation state (about 24 h). The product waspurified on a LH-20 reverse phase column using H₂O as the eluent. Yield:50%.

Example 6 Synthesis of Transition Metal Complexes with Mixed Ligands

Transition metal complexes containing multiple types of ligands can besynthesized stepwise. First, a transition metal complex intermediatethat contains one desired type of ligand and halide ligand(s), forexample, chloride, is synthesized. Then the intermediate is subjected toa ligand substitution reaction to displace the halide ligand(s) withanother desired type of ligand. The preparation of the following osmiumcomplex illustrates the general synthetic scheme.

Synthesis of Compound U: Potassium hexachloroosmate (1 g, 2.08 mmoles),compound K (0.67 g, 4.16 mmoles) and LiCl (1 g, 23.8 mmoles) weresuspended in 40 mL ethylene glycol in a 250 mL three-necked round bottomflask fitted with a reflux condenser. The suspension was degassed withN₂ for 15 min and then stirred under N₂ at 170° C. in an oil bath for7-8 h, resulting in a dark brown solution. The solvent was removed byhigh vacuum distillation at 90-100° C. bath temperature. The gummy solidwas triturated with acetone twice (2×50 mL) and then with H₂O once (50mL). The product was dried at 50° C. under high vacuum for 24 h.

Synthesis of compound W: A suspension of compound U (119 mg, 0.192mmole) and 4-(4-carboxypiperidino)amino-2,2′-bipyridyl (prepared fromcompound E and ethyl isonipecotate using the synthetic methods forcompounds F and G) was made in 10 mL ethylene glycol in a 100 mLthree-necked round bottom flask equipped with a reflux condenser. Thesuspension was degassed with N₂ for 15 min and then stirred under N₂ at130° C. in an oil bath for 24 h. The dark brown solution was cooled toroom temperature and then poured into EtOAc (50 mL). The precipitate wascollected by suction filtration. The dark brown solid thus obtained wascompound W with osmium in a 2+ oxidation state. For ease ofpurification, the osmium 2+ complex was oxidized to an osmium 3+ complexby dissolving the dark brown solid in 20 mL H₂O and stirring thesolution in open air for 24 h. The resulting dark green solution waspoured into a stirred solution of NH₄PF₆ (1 g) in 20 mL H₂O. Theresulting dark green precipitate of[Os(1,1′-dimethyl-2,2′-biimidazole)₂(4-(4-carboxypiperidino)amino-2,2′-bipyridyl)]³⁺3PF₆⁻ was collected by suction filtration and washed with 5 mL H₂O and thendried at 40° C. under high vacuum for 48 h. The counter anion PF₆ ⁻ of[Os(1,1′-dimethyl-2,2′-biimidazole)₂(4-(4-carboxypiperidino)amino-2,2′-bipyridyl)]³⁺3PF₆⁻ was exchanged to the more water soluble chloride anion. A suspensionof the PF₆ ⁻ salt of compound W (150 mg) and Cl⁻ resin (10 mL) in H₂O(20 mL) was stirred for 24 h, at the end of which period all of osmiumcomplex was dissolved. The dark green solution was separated by suctionfiltration and then lyophilized to give compound W.

The present invention should not be considered limited to the particularexamples described above, but rather should be understood to cover allaspects of the invention as fairly set out in the attached claims.Various modifications, equivalent processes, as well as numerousstructures to which the present invention may be applicable will bereadily apparent to those of skill in the art to which the presentinvention is directed upon review of the instant specification.

1.-18. (canceled)
 19. A redox mediator having the formula:

wherein M is iron, cobalt, ruthenium, osmium, or vanadium; L is abidentate ligand comprising at least one imidazole ring; c is an integerselected from −1 to −5 or +1 to +5 indicating a positive or negativecharge; X represents at least one counter ion; d is an integer from 1 to5 representing the number of counter ions, X; and L₁, L₂, L₃ and L₄ areligands, wherein L₁ and L₂ form a second bidentate ligand and whereinthe second bidentate ligand comprises at least one imidazole ring. 20.The redox mediator of claim 19, wherein L is

wherein R₁, R₂, and R′₁ are independently —H or substituted orunsubstituted alkyl, alkenyl, or aryl; and R₃, R₄, R₅, R₆, R′₃, R′₄,R_(a), R_(b), R_(c), and R_(d) are independently —H, —F, —Cl, —Br, —I,—NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, —OH, —NH₂, or substituted orunsubstituted alkoxylcarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkoxy, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido,hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio,alkenyl, aryl, or alkyl.
 21. The redox mediator of claim 20, whereinboth R₁, R₁′, and R₂ are unsubstituted C1 to C12 alkyl.
 22. The redoxmediator of claim 20, wherein R₃, R₄, R₅ and R₆ are —H.
 23. The redoxmediator of claim 19, wherein L₁ comprises a heterocyclic compoundcontaining at least one nitrogen atom.
 24. The redox mediator of claim19, wherein L₁ comprises a heterocyclic compound coupled to a polymericbackbone.
 25. The redox mediator of claim 19, wherein at least one of L,L₁, L₂, L₃ and L₄ is coupled to a polymeric backbone.
 26. The redoxmediator of claim 19, wherein the second bidentate ligand comprises a2,2′-bipyridine having the following formula:

wherein R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, R₂₁, R₂₂ and R₂₃ are independently —H,—F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, —OH, —NH₂, orsubstituted or unsubstituted alkoxylcarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkoxy, alkylamino, dialkylamino, alkanoylamino,arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino,alkylthio, alkenyl, aryl, or alkyl.
 27. The redox mediator of claim 19,wherein L₃ and L₄ in combination form a third bidentate ligand.
 28. Theredox mediator of claim 27, wherein at least one of the second and thirdbidentate ligands is a substituted or unsubstituted 2,2′-bipyridine, asubstituted or unsubstituted 2,2′-biimidazole, or a substituted orunsubstituted 2-(2-pyridyl)imidazole.
 29. The redox mediator of claim27, wherein M is osmium and the transition complex has the followingformula:

wherein R₃, R₄, R₅, R₆, R₁₆, R₁₇, R₁₉, R₂₀, R₂₂ and R₂₃ are —H; R₁ andR₂ are independently substituted or unsubstituted C1 to C12 alkyls; andR₁₈ and R₂₁ are independently —H or substituted or unsubstituted C1-C12alkoxy, C1-12 alkylthio, C1-C12 alkylamino, C2-C24 dialkylamino, orC1-C12 alkyl.
 30. The redox mediator of claim 29, wherein at least oneof R₁, R₂, R₁₈ and R₂₁ comprises a reactive group, wherein the reactivegroup is a carboxy, an activated ester, a sulfonyl halide, a sulfonateester, an isocyanate, an isothiocyanate, an epoxide, an aziridine, ahalide, an aldehyde, a ketone, an amine, an acrylamide, a thiol, an acylazide, an acyl halide, a hydrazine, a hydroxylamine, an alkyl halide, animidazole, a pyridine, a phenol, an alkyl sulfonate, a halotriazine, animido ester, a maleimide, a hydrazide, a hydroxy, or a photo-reactiveazido aryl group.
 31. The redox mediator of claim 29, wherein at leastone of R₁, R₂, R₁₈, and R₂₁ is coupled to a polymeric backbone.
 32. Theredox mediator of claim 27, wherein M is osmium and the transitioncomplex has the following formula:

wherein R₃, R₄, R₅, R₆, R′₃, R′₄, R_(a), and R_(c) are —H; R_(d) is —Hor methyl; R_(b) is —H or substituted or unsubstituted C1-C12 alkoxy,C1-12 alkylthio, C1-C12 alkylamino, C2-C24 dialkylamino, or C1-C12alkyl; and R₁, R′₁ and R₂ are independently substituted or unsubstitutedC1 to C12 alkyl.
 33. The redox mediator of claim 32, wherein at leastone of R₁, R₂, and R′₁ comprises a reactive group, wherein the reactivegroup is a carboxy, an activated ester, a sulfonyl halide, a sulfonateester, an isocyanate, an isothiocyanate, an epoxide, an aziridine, ahalide, an aldehyde, a ketone, an amine, an acrylamide, a thiol, an acylazide, an acyl halide, a hydrazine, a hydroxylamine, an alkyl halide, animidazole, a pyridine, a phenol, an alkyl sulfonate, a halotriazine, animido ester, a maleimide, a hydrazide, a hydroxy, or a photo-reactiveazido aryl group.
 34. The redox mediator of claim 32, wherein at leastone of R₁, R₂, and R′₁ is coupled to a polymeric backbone.